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United States Patent |
5,219,763
|
Van Hoegaerden
|
June 15, 1993
|
Agglutination method for the determination of multiple ligands
Abstract
The invention provides a method capable of determining the presence or
absence of each of a plurality of different ligands in a specimen. The
specimen is contacted with a predetermined number of different homogenous
populations of fluorescent beads having one or more predetermined
antiligands affixed to their surface. The specimen and bead mixture is
analyzed using a means having a single parameter of measuring fluorescence
per ligand to determine the number of non-agglutinated beads, the number
of agglutinated beads, the number of bead aggregates, and for each
aggregate, the number of beads its comprises. This information is used to
correlate the presence or absence in the specimen of each of the different
ligands analyzed for. The method of the invention thus provides for the
simultaneous determination of a predetermined number of ligands in a
specimen using only a single bead contacting step.
Inventors:
|
Van Hoegaerden; Michel (Mormant, FR)
|
Assignee:
|
Chemunex (Maisons Alfort, FR)
|
Appl. No.:
|
499509 |
Filed:
|
August 3, 1990 |
PCT Filed:
|
November 3, 1989
|
PCT NO:
|
PCT/FR89/00573
|
371 Date:
|
August 3, 1990
|
102(e) Date:
|
August 3, 1990
|
PCT PUB.NO.:
|
WO90/05307 |
PCT PUB. Date:
|
May 17, 1990 |
Foreign Application Priority Data
Current U.S. Class: |
436/523; 435/973; 436/524; 436/528; 436/531; 436/534; 436/536; 436/538; 436/800; 436/805 |
Intern'l Class: |
G01N 033/538; G01N 033/546 |
Field of Search: |
422/82.01,82.08
436/523,524,527-531,533,534,538,536,800,805
435/973
|
References Cited
U.S. Patent Documents
4499052 | Feb., 1985 | Fulwyler | 422/82.
|
4550017 | Oct., 1985 | Liu et al. | 436/520.
|
4745075 | May., 1988 | Hadfield et al. | 436/531.
|
4829011 | May., 1989 | Gibbons | 436/533.
|
4837168 | Jun., 1989 | de Jaeger et al. | 436/800.
|
4918004 | Apr., 1990 | Schwartz | 436/800.
|
4934811 | Jun., 1990 | Watts et al. | 356/73.
|
Foreign Patent Documents |
126450 | Nov., 1984 | EP.
| |
3811566 | Oct., 1988 | DE.
| |
2627286 | Aug., 1989 | FR.
| |
Other References
Patent Abstracts of Japan, vol. 10, No. 322, Oct. 31, 1986, p. 511, JP-A-61
128169, Jun. 16, 1986, Mitsubishi Chem. Ind. Ltd.
Biological Abstracts, vol. 82, No. 5, 1986, P. K. Bjornsen: "Automatic
Determination of Bacterioplankton Biomass by Image Analysis", p. AB-574,
Abstract 45417.
|
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Chin; Christopher L.
Attorney, Agent or Firm: Bell, Seltzer, Park & Gibson
Claims
I claim:
1. A method for determining the presence or absence of each of a
predetermined number of three or more ligands in a liquid, semi-liquid or
pasty specimen, by a direct or indirect aggultination, or an aggultination
inhibition method by competition, comprising the steps:
contacting the specimen with a predetermined number of homogenous
populations of fluorescent beads having a specific fluorescence and one or
more pre-determined antiligands fixed to their surface, said predetermined
number of populations being less than said predetermined number of ligands
and wherein at least one of said ligands is capable of binding to the
antiligand on two or more of said homogeneous populations of beads;
analyzing said specimen containing said beads using a measuring means
capable of discriminating between the specific fluorescence of said beads
for each ligand;
determining in response to said analyzing step the number of
non-agglutinated beads, the number of agglutinated beads, the number of
bead aggregates and for each aggregate, the number of the beads it
comprises; and
correlating the information determined in said determining step with the
presence or absence in said specimen, of each of said different ligands to
thereby enable the simultaneous determination of a plurality of ligands
using a single bead contacting step.
2. The method according to claim 1, wherein said beads comprise at least
two predetermined antiligands fixed to their surface.
3. The method according to claim 1, wherein said beads have a diameter of
between 0.05 .mu.m and 10 .mu.m.
4. The method according to claim 1, wherein said antiligand is selected
from the group consisting of antigens, antibodies, receptors, lectins,
proteins of type A or G, and avidin, said antiligand being specific to one
or more of said predetermined number of different ligands.
5. The method according to claim 4, wherein the antibodies are monoclonal
antibodies.
6. The method according to claim 4, wherein the antibodies are polyclonal
antibodies.
7. The method according to claim 1, wherein said measuring means used in
said analyzing step is selected from the group consisting of a flow
cytometer, an image analyzer and a laser sweep system.
8. The method according to claim 1 wherein said correlating step is
conducted in a manner to determine quantitative values for each of said
ligands.
9. A method for determining the presence or absence of each of a
predetermined number of a plurality of different ligands in a liquid,
semi-liquid or pasty specimen, by a direct or indirect agglutination, or
an agglutination inhibition method by competition, comprising the steps:
contacting the specimen with a predetermined plurality of homogenous
populations of fluorescent beads having a specific fluorescence and one or
more predetermined antiligands fixed to their surface, said predetermined
plurality of populations being less than said predetermined number of
ligands and wherein at least one of said ligands is capable of binding to
the antiligand on two or more of said homogeneous populations of beads;
when said predetermined number of ligands;
analyzing said specimen containing said beads using a measuring means
capable of discriminating between the specific fluorescence of said beads
for each ligand;
determining in response to said analyzing step, the number of
non-agglutinated beads, the number of agglutinated beads, the number of
bead aggregates and for each aggregate, the number of beads it comprises
and the population type or types of beads it comprises; and
correlating the information determined in said determining step with the
presence or absence in said specimen of each of said different ligands to
thereby enable the simultaneous determination of a plurality of ligands
using a single bead contacting step.
10. The method according to claim 9 wherein the beads in at least one of
said homogenous populations of beads comprise at least two antiligands
affixed to their surface.
11. The method according to claim 9 wherein said beads have a diameter of
between 0.05 .mu.m and 10 .mu.m.
12. The method according to claim 9 wherein the antiligands fixed to the
surface of said beads are specific to one or more of said predetermined
plurality of different ligands and are selected from the group consisting
of antigens, antibodies, receptors, lectins, proteins of type A or G, or
avidin.
13. The method according to claim 12 wherein the antibodies are monoclonal
antibodies.
14. The method according to claim 12 wherein the antibodies are polyclonal
antibodies.
15. The method according to claim 9 wherein the measuring means used in
said analyzing step is selected from the group consisting of a flow
cytometer, an image analyzer and a laser sweep system.
16. The method according to claim 9 wherein said correlating step is
conducted in a manner to determine quantitative values for each of said
ligands.
Description
The present invention relates to a process for accurately and specifically
determining in a liquid or semi-liquid, at least one biological, organic
or drug soluble substance, even if it is present in extremely small
amounts by means of a suitable agglutination reaction.
It is known to detect and to determine soluble substances and particularly
peptides or proteins by an agglutination reaction or an agglutination
inhibition reaction.
The agglutination reaction is the direct consequence of the fixation of an
agglutinating antibody on a cell, for example, and thus calls for
antigen-antibody complexes or hormone receptor complexes having
agglutinating properties.
Nonetheless, it should be recalled that although these reactions may be
easy to produce, their interpretation by the naked eye is mostly delicate
and requires the presence of a large amount of substance to be assayed in
the specimen, for interpretation to be easy.
An agglutination reaction can be contemplated in various biological
contexts; in particular in the field of immunology, agglutination is the
manifestation of the formation of a complex.
There exist so-called direct methods (antigen/antibody complex), so-called
indirect methods or methods of inhibition by competition; there are also
known assay methods of the so-called "sandwich" or bisite type; in these
"sandwich" type methods, two antibodies are used, one to capture the
antigen, the other to reveal its presence, if this antigen is in fact
present and has been captured by the first antibody. Generally, the first
antibody is fixed to a solid support (plate, bead . . . ), the second
antibody being conjugated with a reaction "developer" such as
fluorochrome, radio-isotope, or enzyme.
In an agglutination reaction, red blood cells coated with an antigen serve
for detecting corresponding specific antibodies (hemagglutination). An
extension of the agglutination methods has consisted of using plastic
beads: in the case of a positive reaction, the appearance of a macroscopic
"granulation" (passive agglutination) is observed. A reverse procedure has
also been described: the antibodies are fixed to beads and serve to detect
the corresponding antigens. The sensitivity of such a method is high, but,
as has already been mentioned above, the interpretation of the result is
not objective and is variable as a function of the observer, the amount of
agglutination being a function of the amount of antigen present for a
certain range of concentrations of this antigen.
In order to overcome this drawback of agglutination methods and in order to
make them quantitative, a certain number of authors have proposed the
counting of the non-agglutinated particles by means of a suitable particle
counter especially a blood cell counter.
MASSON et al (Particle Counting Immunoassay, Meth. Enzymol., 1981, 74,
106-139) propose a method of determining various antigens (proteins,
peptides . . . ), antibodies, haptens and immunocomplexes by means of an
agglutination reaction between latex particles 0.8 .mu.m in diameter
coated with antibodies or suitable antigens and said antigens or
antibodies or haptens or immunocomplexes, and the counting of the
non-agglutinated particles by means of a particle counter, particularly a
red blood cell counter which is a small angle defracted light detector.
The counting of the non-agglutinated particles enables evaluation of the
extent of the agglutination reaction and, consequently, the amount of
product to be determined.
The detector is parametered so as to take no account electronically of
particles whose diameter is less than 0.6 .mu.m and particles whose
diameter is greater than 1.2 .mu.m (agglutinated particles).
MASSON described various elements of this method in a certain number of
patents:
U.S. Pat. No. 4,062,935 (1977), claims a method of detecting
antigen/antibody complexes which comprises the addition to the sample of a
rheumatoid factor (RF) and material which agglutinates RF on contact, then
detection of the amount of agglutination of said material compared with a
standard mixture of said material and of RF; said material is constituted
by inert particles coated with immunoglobulins, said particles being, more
particularly, of polystyrene. This patent also describes a method of
detection of the presence or the absence in a specimen of biological fluid
of antibody/antigen complexes comprising the addition to the sample of a
known amount of RF and a known amount of latex particles coated with IgG's
characterised in that the RF is bound to any antigen/antibody complex
present, the remainder of the RF entraining the agglutination of latex
particles; then determination of the amount of said RF remainder by
counting agglutinated latex particles and comparison of the result with a
standard graph.
U.S. Pat. No. 4,138,213 (1979) describes an automated method of determing a
quantity of an antibody or an antigen in a biological fluid specimen,
comprising several steps and particularly a step of counting the number of
non-agglutinated particles.
U.S. Pat. No. 4,162,895 (1979) describes a method of detecting antigens,
antibodies or antigen/antibody complexes, which employes a reaction based
on mouse serum; this reagent is combined only with antigen/antibody
complexes and is used within the scope of agglutination reactions, for
example a method of detecting antibodies which comprises the addition to
the specimen of a known amount of particles coated with antigens
corresponding to said antibodies then counting the number of
non-agglutinated particles and calculation of the amount of antibodies
present in the specimen.
U.S. Pat. No. 4,184,849 (1980) describes a method of detecting antigens and
antibodies in a liquid specimen by employing a suitable agglutination
reaction and detection of the extent of the agglutination, which permits
determination of the amount of antibodies or of antigens present in the
specimen; into the liquid are introduced two reagents in the form of
particles which agglutinate together but whose agglutination is inhibited
by antibodies or antigens possibly present.
U.S. Pat. No. 4,397,960 (1983) describes an assay method for an antigen by
an agglutination reaction. The assay consists of the determination of the
extent of agglutination of latex particles in the solution and thus
permits the determination of the amount of antigen in the specimen; the
estimation of the agglutination is performed by selective counting of the
agglutinated and non-agglutinated latex particles.
These various methods have nonetheless a certain number of drawbacks:
it is necessary to treat the specimen so as to remove any particle which
could constitute a false positive; to do this, it is necessary to subject
the specimen to a certain number of physical or chemical operations such
as the use of particular buffers, filtration, centrifugation;
the numbering of the agglutinated particles is only possible in clear
media;
besides, counting by small angle defraction does not permit good
sensitivity if it attempts to count agglutinated particles (unseparated
peaks and hence difficult to interpret);
in addition, they do not permit the simultaneous detection of several
ligands.
Applicant consequently made it an object to provide a method not having the
drawbacks of the agglutination methods described in the prior art.
By antiligand is meant a molecular structure capable of recognizing and
binding specifically to the one or more organic, biological or medicinal
substances to be sought, called below ligands.
It is an object of the present invention to provide a specific method of
detecting and/or of determining one or several ligands to be analyzed in a
liquid, semi-liquid or pasty specimen, by a direct, indirect agglutination
method or agglutination inhibition method, by competion, by employing
fluorescent structures, called below beads, adapted to fix a suitable
antiligand, which method is characterized in that the specimen to be
analyzed is contacted with a single homogeneous population of beads per
ligand up to two ligands to be detected and less than one population of
beads per ligand for three ligands and more to be detected and wherein
there is detected and/or counted simultaneously the number of
non-agglutinated beads, the number of agglutinated beads, the number of
bead aggregates, and for each aggregate, the number of beads that it
comprises, using measuring means with a single parameter that is, a unique
or different parameter, of measuring fluorescence for each ligand, said
method enabling, by the single contacting, the detection and/or the
determination of several ligands simultaneously.
By homogeneous population of fluorescent beads, is meant a collection of
identical beads, that is to say whose fluorochrome emission wavelength,
diameter and/or density are identical, as well as the one or more
antiligands fixed to their surface.
According to an advantageous embodiment of the method according to the
invention, said beads comprise at least two antiligands fixed to their
surface.
According to another advantageous embodiment of the method according to the
invention, said beads have a diameter comprised between 0.05 .mu.m and
10.0 .mu.m.
The fluorescent beads according to the detection system can be,
particularly, latex beads, polyacrylamide beads, cellulose beads,
polystyrene beads, agarose beads, PVC beads or glass beads.
According to yet another advantageous embodiment of the method according to
the invention, the one or more antiligands are selected from the group
which comprises antigens, antibodies, receptors, lectins, proteins for
fixation to immunoglobulins of type A or G and avidines, specific to the
one or more substances or ligands to be sought.
According to an advantageous feature of this embodiment, the antibodies are
monoclonal antibodies.
According to another advantageous feature of this embodiment, the
antibodies are polyclonal antibodies.
The total amount of fluorescent light and defracted lights vary as a
function of the number of agglutinated beads in each agregate or
agglutinate and thus enable, using detection means for fluorescent and/or
defracted light, the discrimination of the number of fluorescent beads
present in each agglutinate and of their size.
Such discrimination enables the number of doublets, triplets, quadruplets
etc., formed to be known and gives, for this reason, to this method of
determination, a sensitivity and a precision of measurement which are
distinctly increased.
The substances to be analyzed which can particularly be detected by the
method according to the invention, are all ligands capable of fixing an
antiligand, for example organic, biological or medicinal substances and
especially antigens, antibodies, haptens and immuno-complexes, hormones,
membranal receptors, lectins, proteins of the type A or G or avidines.
In yet another embodiment of the method according to the invention, the
fluorescence measuring means is selected from the group which comprises
the flow cytometer, the image analyzer and a laser sweep system.
The method of detection and of counting according to the invention, has a
certain number of advantages both economic (little manipulation, very fast
counting time) and technical:
it permits, particularly, the counting of agglutinated and non-agglutinated
beads in a turbid or viscous medium;
it permits the counting of agglutinated and non-agglutinated beads almost
singly;
it is, for this reason, very sensitive, which enables the determination of
very small amounts of a desired substance;
the fluorescent beads used avoid interference with other particles possibly
present and oblige neither a physical separation of particles possibly
present in the specimen to be assayed nor washing steps, which constitutes
a homogeneous assay; in fact, the use of the detection system according to
the invention permits working in a complex medium containing a large mass
of other particles, the number of self-fluorescent elements being lower
and can be ignored by the detection means, especially by a judicious
choice of optical filters;
it enables discrimination of non-agglutinated beads, doublets, triplets,
etc . . . , since it permits an absolute measurement of the intensity and
the fluoresence wave-length of each particle or aggregate of particles,
one by one.
it is possible, especially, and this, without this being limiting, to use
the flow cytometer described in French Patent application no. 88 02937: it
permits the handling of specimens of considerable volume, possesses an
automatic washing system between each sample, and is designed for routine
use; it is, in addition, capable of detecting each agglutinated or
non-agglutinated bead and of counting them individually, up to a frequency
of 3,000 per second.
The form of the flow cell is selected to reduce to their minimum
hydrodynamic extraction forces.
It is in addition an object of the present invention to provide a kit or
outfit ready-for-use for practising the detection and/or assay method
according to the invention, characterized in that it comprises at least
one population of beads on which is fixed at least one suitable
antiligand, possibly associated with suitable buffers.
According to an advantageous embodiment of the kit according to the
invention, it comprises at least one bead population, on which are fixed
at least two suitable different antiligands.
In another advantageous embodiment of the kit according to the invention,
it comprises different populations of beads in admixture.
Besides the foregoing features, the invention comprises yet other features
which will emerge from the description which follows, which refers to
examples of practising the method according to the invention.
It must be well-understood, however, that these examples are given purely
by way of illustration of the invention, of which they do not constitute
in any way a limitation.
The method according to the invention comprises the formation of complexes
by contact of the specimen with fluorescent beads on which is fixed at
least one suitable antiligand, under conditions suitable for the
appearance of an agglutination or for the inhibition of an agglutination,
and then the detection of the agglutinated and non-agglutinated beads.
In a preferred embodiment of the method, populations of fluorescent beads
bearing complementary antiligands of the ligand to be assayed are mixed in
equal number, about 50,000 per ml. The solution or suspension to be tested
is added in a volume which can vary from 3 to 1,000 .mu.l or more. An
incubation of 37.degree. C. accelerates the formation of the
ligand-antiligand bond; the duration of this incubation depends
essentially on the affinity constant of the antiligands employed; the
final sensitivity of the test also will depend thereon.
The method according to the invention, permits the detection of a single
fluorescent bead. If two of them are counted, it is possible
simultaneously to determine if they are separated or agglutinated, and in
the case where they are agglutinated, if they have the same
characteristics or not. The sensitivity of this method thus enables,
theoretically, measurement of the presence of a single ligand molecule, if
the latter succeeds in agglutinating two fluorescent beads, of the same
spectral characteristics or not.
In another advantageous embodiment of the method, the measurement of a
concentration of small molecules, to which two antiligands could not be
fixed is also possible: the fixation of the ligand (haptens) or of a
derivative of the latter on a bead population of the specific antiligand
on another bead population enables the performance of a test in
competition in which the agglutination of the beads is inhibited by the
free hapten or a derivative of the latter, possibly present to be assayed.
EXAMPLE 1
Detection of enterotoxin A (A-type protein) of Staphylococcus aureus by the
method according to the invention
1) Preparation of fluorescent beads coated with an antiligand (detection
system)
In a first phase, the detection system is prepared by coupling fluorescent
particles with the appropriate reagent by known methods.
Covalent coupling of carboxylated fluorescent particles with
anti-enterotoxin A rabbit polyclonal antibodies:
a--Preparation of beads (Fluoresbrite-Polysciences):
0.5 mg of beads in 2.5% suspension in 4 ml of borate buffer (10.sup.-2 M
boric acid+0.15M NaCl, pH 8.1);
prepare a solution containing 80 .mu.moles of hexamethylene-diamine in 1 ml
of borate buffer;
add thereto the 4 ml of beads;
add 0.05M carbodiimide;
incubate overnight at 4.degree. C.;
wash with the borate buffer.
b--Preparation of the antibody:
dialyse the antibodies against phosphate buffer 0.1M, pH 6;
provide 2/3 antibodies per 1/3 of beads;
add 200 .mu.l of NaIO.sub.4 0.15M;
incubate 3 hours at 4.degree. C. in darkness;
add glycerol to a final concentration of 0.015M;
incubate 30 minutes at ambient temperature;
dialyse against phosphate buffer pH 6;
c--Coupling proper:
add the beads and the antibodies in the presence of 5 ml of borate buffer;
incubate 16 hours at 4.degree. C. with stirring;
adjust the pH to 8.8;
stabilise with NaBH.sub.4 (5 ml in the test);
incubate 5 hours at 4.degree. C.;
wash with 0.5% PBS-BSA-0.1% Tween.
Non-covalent coupling of carboxylated fluorescent particles with
anti-enterotoxin A rabbit polyclonal antibodies:
a--In a glass tube:
100 .mu.l of an antibody solution with 2.5 mg/ml;
100 .mu.l of Glycine buffer (Meth. Enzymol. 74, 111);
5 .mu.l of beads diluted to 1/10;
b--Stirring for 30 minutes at ambiant temperature;
c--Rinsing: 3 times in a 0.5% PBS-BSA-0.1% Tween buffer;
d--Take up again in 100 .mu.l PBS.
2) Detection of the substance sought:
a) a detection system such as prepared in 1) is used;
b) the detection system is mixed with a quantity of antigen; it is
incubated 2 hours at 37.degree. C. and the agglutinated and
non-agglutinated beads are detected; the results are presented in Table I
and in FIG. 1.
Table I, below, shows a comparative test between a negative control (beads
coated with antibodies but absence of enterotoxin) for which 10.5% of
agglutinated beads are obtained (FIG. 1 and column A of Table I) and
a detection of enterotoxin A, by the method according to the invention,
(FIG. 2 and column B of Table I), for which 85% of agglutinated beads are
obtained, in the presence of a specimen containing 10 mg of enterotoxin A;
the agglutination is proportional to the concentration of enterotoxin A
within a given range of concentrations.
In FIGS. 1, 2 and 3, are shown as abscissae, the relative fluorescence
measurement (channel) the number of the channel and as ordinate, the
number of particles or of aggregates of particles per channel.
FIG. 1 shows the result obtained with latex beads coupled to antibodies
(negative control) and corresponds to the results mentioned in Table I,
column A.
FIG. 2 shows, in the form of a histogram, the results mentioned in Table I,
column B.
FIG. 3 shows the result obtained with fluorescent latex beads not coupled
to antibodies (other negative control) but in the presence of antigen. An
agglutination of less than 2% is observed.
TABLE I
__________________________________________________________________________
CARBOXYLATED 1.1 .mu.m BEADS COUPLED WITH AN ANTI-ENTEROTOXIN A
RABBIT POLYCLONAL ANTIBODY
1.1 .mu.m BEADS + ANTIBODY +
1.1 .mu.m BEADS + ANTIBODY A
10 ng ENTEROTOXIN A
No of beads A B
per clump
Channel
Event/Histo
Beads/Histo
% Event/Histo
Beads/Histo
%
__________________________________________________________________________
1 5225
9475 9475 89,522
8312 8312 15,497
2 1125
466 932 8,8057
4198 8396 15,654
3 16875
59 177 1,6723
2714 8142 15,18
4 225 1581 6324 11,791
5 28125 1145 5725 10,674
6 3375 660 3960 7,3832
7 39375 495 3465 6,4603
8 450 245 1960 3,6543
9 50625 180 1620 3,0204
10 5625 126 1260 2,3492
11 61875 122 1342 2,5021
12 675 69 828 1,5438
13 73125 58 754 1,4058
14 7875 33 462 0,8614
15 84375 31 465 0,867
16 900 11 176 0,3281
17 95625 12 204 0,3803
18 10125 13 240 0,4475
19 10688 13 240 0,4475
SUM 10000 10584 100 20005 53635 100
__________________________________________________________________________
Table II, below, shows the results obtained with a negative control (beads
coated with antibodies, but in the absence of corresponding antigen) under
slightly different operational conditions (gentle stirring for 30 minutes
only). In this case, 1.6% of agglutinated beads are obtained (background
noise and interference very slight).
TABLE II
______________________________________
1.1 .mu.m BEADS + ANTIBODIES
Number 30 MIN
of beads Event/ Beads/
per clump Channel Histo Histo %
______________________________________
1 56 10787 10787 98.395
2 112 88 176 1.6054
3 168
4 224
5 280
6 336
7 392
8 448
9 504
10 560
11 616
12 672
13 728
14 784
15 840
16 896
17 952
18 1008
19
SUM 10875 10963 100
______________________________________
EXAMPLE 2
Study of the stability of beads coated with suitable antibodies
Beads coated with anti-enterotoxin A rabbit polyclonal antibodies, such as
described in Example 1, are used one week after their preparation; the
results as shown in Table III are obtained:
in column A are shown the results relating to a negative control, beads
coated with antibodies;
in column B are shown the results concerning an enterotoxin A detection
test, the amount of the latter being 10 ng; the reading of the result is
performed after 20 minutes with gentle stirring;
in column C are shown the results relating to an enterotoxin A detection
test, the amount of the latter being 50 ng; the reading of the result is
carried out after 20 minutes with gentle stirring;
an inhibition of the agglutination of the beads in the case of excess
antigen (50 ng against 10 ng) is observed: 51.3% of non-agglutinated beads
against 42.7% of non-agglutinated beads is observed; the addition of free
specific antibodies re-establishes the agglutination of the beads coated
with antibodies which are saturated with antigen.
TABLE III
__________________________________________________________________________
CARBOXYLATED 1.1 .mu.m BEADS COUPLED WITH AN ANTI-ENTEROTOXIN A
RABBIT POLYCLONAL ANTIBODY
B C
Number A 1.1 .mu.m BEADS + 1.1 .mu.m BEADS +
of beads 1.1 .mu.m BEADS + ANTIBODIES + 10 ng
ANTIBODIES + 50 ng
per Chan-
ANTIBODIES ENTEROTOXIN A 20 min
ENTEROTOXIN A 20 min
clump
nel Event/Histo
Beads/Histo
% Event/Histo
Beads/Histo
% Event/Histo
Beads/Histo
%
__________________________________________________________________________
1 56 7831 7831 92.119
11308 11308 42.7
11878 11878 51.29
2 112 158 316 3.717
2280 4560 17.22
1714 3428 14.80
3 168 118 354 4.164
777 2331 8.80
663 1989 8.58
4 224 436 1744 6.58
313 1252 5.40
5 280 239 1195 4.51
196 980 4.23
6 336 163 978 3.69
113 678 2.92
7 392 127 889 3.35
68 476 2.05
8 448 80 640 2.41
52 416 1.79
9 504 72 648 2.44
44 396 1.71
10 560 41 410 1.54
30 300 1.29
11 616 41 451 1.70
26 286 1.23
12 672 26 312 1.17
22 264 1.14
13 728 16 208 0.78
12 156 0.67
14 784 18 252 0.95
10 140 0.60
15 840 9 135 0.51
10 150 0.64
16 896 8 128 0.48
11 176 0.76
17 952 14 238 0.89
8 136 0.58
18 1008 3 54 0.20
3 54 0.23
SUM 8107 8501 100 15658 26481 100 15173 23155 100
__________________________________________________________________________
In the following examples, by green fluorescent beads is meant beads marked
with coumarin and by red fluorescent beads, beads marked with
phycoerythrin R.
EXAMPLE 3
Analysis of the immune protection of the pregnant woman against
toxoplasmosis
Fluorescent beads coated with toxoplasmic antigen as well as fluorescent
beads coated with anti-IgG antibodies and beads covered with anti-IgM
antibodies as in Example 1 (1a) are prepared.
The objective consisted of seeking simultaneously the presence of
anti-toxoplasmin antibodies of isotypes IgG and IgM. Green fluorescent
beads were coated with toxoplasmic antigen and were then incubated with
the blood specimen in the presence of green beads coated with anti-IgG
antibodies and with red beads coated with anti-IgM antibodies. The
proportion of green/green doublets on the one hand and green/red on the
other hand enables the searching and quantifying simultaneously of IgG and
IgM antibodies. The aggregates with three beads or more were analyzed for
their red and green components. In addition, a reference curve was
established.
EXAMPLE 4
Rapid determination of isomers or of denaturated epitopes
Fluorescent beads coated with a suitable antiligand as in Example 1 are
prepared.
A monoclonal antibody common to all isomers or recognising all native and
denaturated forms of a protein coats green beads. A specific monoclonal
antibody of each modified form is coupled to beads either green or red.
The search for green/green and green/red aggregates gives simultaneously
the total amount of antigen and the proportion of two (or more, if several
bead colours are selected) forms of the molecule.
EXAMPLE 5
Determination of haptens by inhibition of agglutination
Beads coated with the appropriate antiligand are prepared as in Example 1.
Fluorescent beads are coated with a derivative of the hapten, for example
aflatoxin-poly-L-lysine. The mixture of these beads with a homogeneous
population of beads coated with an anti-aflatoxin monoclonal antibody will
give agglutination. The possible presence of free aflatoxin in a food
specimen will cause, on its addition to the mixture of beads, an
inhibition of agglutination by competition in its fixation to the
monoclonal antibody.
EXAMPLE 6
Search, analysis and determination of complex carbohydrates
The fixation of lectins of different specificities (for example
concanavaline A for .alpha.-D-glucosyl and agglutinin of wheat germ for
[.beta.-N-acetylglucosaminyl].sub.n or sialic acids) on beads of different
colors, according to the procedure of Example 1, enables the determination
of whether the sugars recognized by these lectins are borne by the same
molecule (agglutination) or by different molecules (no agglutination);
this constitutes also a tool for the determination of structures: there
are known to date in fact several tens of different lectins.
EXAMPLE 7
Search for very low concentrations of antibodies (amplification of
agglutination)
A very small amount of antibody cannot cause spontaneous agglutination.
This is the case in the determination of specific IgG's in allergy
diagnosis.
Different allergens are coupled with beads of different sizes and/or
colors, according to the procedure of Example 1. The mixture of this bead
population with the biological specimen will cause the fixation of the
specific antibodies to the corresponding allergens. Amplification of
agglutination is then obtained by adding a bivalent specific anti-IgE
antibody which will associate two IgE molecules and hence two beads.
This test can also be used for the search for and determination of IgM, IgA
and IgD.
EXAMPLE 8
Search for specific sequences of nucleic acids
Two (or more) nucleic probes hybridizing with complementary sequences on a
same gene are coupled to beads of different colors, according to the
procedure of Example 1.
The nucleic material, freed from the cells, is placed in the presence of
different populations of fluorescent beads under hybridization conditions.
The search for aggregates is done by flow cytometry, by image analysis or
by laser sweep. The presence of aggregates constituted from beads of the
same color indicates the presence of repetitive sequences on the same gene
or the same chromosome whereas aggregates constituted from beads of
different colors indicates the complementary distance of two (or more) of
the specific sequences sought.
Thus as is apparent from the foregoing, the invention is in no way limited
to those of its types of practice, embodiments and uses which have just
been described more explicitly; it encompasses, on the contrary, all
modifications which may come to the spirit of the technician skilled in
the art, without departing from either the framework, or the scope of the
present invention.
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